Quantifying transient 3D dynamical phenomena of single mRNA particles in live yeast cell measurements.

Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4 ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions.

Widespread mRNA association with cytoskeletal motor proteins and identification and dynamics of myosin-associated mRNAs in S. cerevisiae.

Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe here a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP.

Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function.

Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit.

Communication between levels of transcriptional control improves robustness and adaptivity.

Regulation of eukaryotic gene expression depends on groups of related proteins acting at the levels of chromatin organization, transcriptional initiation, RNA processing, and nuclear transport. However, a unified understanding of how these different levels of transcriptional control interact has been lacking. Here, we combine genome-wide protein-DNA binding data from multiple sources to infer the connections between functional groups of regulators in Saccharomyces cerevisiae.

Genomic association of the proteasome demonstrates overlapping gene regulatory activity with transcription factor substrates.

The proteasome can regulate transcription through proteolytic processing of transcription factors and via gene locus binding, but few targets of proteasomal regulation have been identified. Using genome-wide location analysis and transcriptional profiling in Saccharomyces cerevisiae, we have established which genes are bound and regulated by the proteasome and by Spt23 and Mga2, transcription factors activated by the proteasome. We observed proteasome association with gene sets that are highly transcribed, controlled by the mating type loci, and involved in lipid metabolism.